Cell junction proteins in podocyte injury repair

描述（由申请人提供）：成人肾足细胞的主要细胞连接是狭缝隔膜，它是肾脏滤过屏障的重要组成部分。足突消失过程中狭缝隔膜的丧失导致蛋白尿，并导致许多慢性肾脏疾病的进展，如糖尿病和hiv相关肾病。足细胞细胞连接的发育形成和成体稳态是一个尚未完全了解的过程，但对于修复或替换在疾病中受损或丢失的足细胞的治疗策略的发展是重要的。我们最近发现了cladin样细胞连接蛋白TM4SF10和Fyn结合蛋白ADAP，它们是足细胞表达的影响板足形成、Fyn活性和Nephrin磷酸化的蛋白。TM4SF10在足细胞发育过程中短暂表达，在损伤修复过程中重新表达。因此，我们假设在疾病中，TM4SF10可能通过保留简化的细胞连接和修改Fyn的活性来保护足突消退过程中的足细胞。我们还假设，ADAP是一种已知在整合素信号传导中起作用的大型适配蛋白，在足细胞足突的缝隙隔膜和足突的整合素附着物处整合肌动蛋白细胞骨架动力学中起支架中心的作用。我们将通过使用标准突变分析和体外结合试验来确定ADAP与TM4SF10和其他狭缝隔膜复合物的关键细胞内组分的结合，并基于板足延伸、整合素结合和Nephrin磷酸化来评估功能影响，从而验证这一假设。拟议的研究还将包括进一步表征ADAP基因敲除小鼠的肾脏表型，以及进一步开发和表征TM4SF10基因敲除小鼠模型。将建立这些小鼠的足细胞细胞系，以研究TM4SF10和ADAP在足细胞中调节整合素细胞附着和Nephrin磷酸化的必要功能。此外，我们已经确定TM4SF10的功能可以直接抑制Fyn活性，可能是通过将Fyn从质膜分离出来。这将通过实时成像和共定位方法来研究，评估在TM4SF10存在和不存在的情况下Fyn的细胞内运输。这些研究描述了第一个完整的分子机制，将细胞-细胞在狭缝隔膜上的粘附与细胞-基质粘附以及足突中整合素的结合联系起来，这些事件需要在足突形成和缩回过程中同步进行。这些研究将对开发针对以足突消退为特征的肾小球疾病的新疗法具有重要意义，其中预防或修复受损的足细胞将是有益的。

DESCRIPTION (provided by applicant): The major cell-cell junction of the adult kidney podocyte is the slit diaphragm, an important component of the kidney filtration barrier. The loss of the slit diaphragm during foot process effacement results in proteinuria and contributes to the progression of many chronic kidney diseases such as diabetes and HIV-associated nephropathy. The developmental formation and adult homeostasis of podocyte cell junctions are processes that are not fully understood, but are important for the development of therapeutic strategies to repair or replace podocytes that are damage or lost in disease. We have recently identified the claudin-like cell junction protein TM4SF10 and the Fyn binding protein ADAP as podocyte-expressed proteins that affect lamellipodia formation, Fyn activity, and Nephrin phosphorylation. TM4SF10 is transiently expressed during podocyte development and is re-expressed during injury repair. Thus, we hypothesize that in disease, TM4SF10 may function to protect the podocyte during foot process effacement by preserving a simplified cell junction and by modifying the activities of Fyn. We also hypothesize that ADAP, a large adapter protein with known roles in integrin signaling, functions as a scaffolding center in the podocyte foot process integrating actin cytoskeleton dynamics at both the slit diaphragm and in integrin attachments of the foot process. We will test this hypothesis by determining the association of ADAP binding to TM4SF10 and other key intracellular components of the slit diaphragm complex using standard mutational analysis and in vitro binding assays, and assess functional effects based on lamellipodia extension, integrin binding, and Nephrin phosphorylation. Proposed studies also will include further characterize of the renal phenotype in ADAP knockout mice and further development and characterization of a knockout mouse model for TM4SF10. Podocyte cells lines from these mice will be established to investigate the requisite functions of TM4SF10 and ADAP in podocytes with regard to regulating integrin cell attachments and Nephrin phosphorylation. In addition, we have established TM4SF10 functions to directly suppress Fyn activity, possibly by sequestering Fyn away from the plasma membrane. This will be investigated with real time imaging and co-localization methods assessing intracellular trafficking of Fyn in the presence and absence of TM4SF10. These studies describe the first integrated molecular mechanism connecting cell-cell adhesion at the slit diaphragm with cell-matrix adhesion with integrin binding in the foot process, events that require synchronization during foot process formation and retraction. These studies will have important implications toward the development of novel therapies directed at glomerular diseases characterized by foot processes effacement where preventing or repairing damaged podocytes would be beneficial.